Substrate preference and phosphatidylinositol
monophosphate inhibition of the catalytic domain of the
Per-Arnt-Sim domain kinase PASKIN
Philipp Schla
¨
fli*, Juliane Tro
¨
ger*,, Katrin Eckhardtà, Emanuela Borter§, Patrick Spielmann and
Roland H. Wenger
Institute of Physiology and Zu
¨
rich Center for Integrative Human Physiology, University of Zu
¨
rich, Switzerland
Keywords
metabolism; phospholipid; protein
translation; ribosomal protein S6;
sensory kinase
Correspondence
R. H. Wenger, Institute of
Physiology, University of Zu
¨
rich,
Winterthurerstrasse 190, CH-8057
Zu
¨
rich, Switzerland
Fax: +41 (0) 44 6356814
Tel: +41 (0) 44 6355065
E-mail:
Website:

monophosphorylated phosphatidylinositols. However, stimulated PASKIN
autophosphorylation did not correlate with ribosomal protein S6 and
eukaryotic elongation factor 1A1 target phosphorylation. Although auto-
phosphorylation was enhanced by monophosphorylated phosphat-
idylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited
autophosphorylation. By contrast, target phosphorylation was always
inhibited, with the highest efficiency for di- and tri-phosphorylated phos-
phatidylinositols. Because phosphatidylinositol monophosphates were
found to interact with the kinase rather than with the PAS domain, these
data suggest a multiligand regulation of PASKIN activity, including a still
unknown PAS domain binding ⁄ activating ligand and kinase domain bind-
ing modulatory phosphatidylinositol phosphates.
Structured digital abstract
l
A list of the large number of protein-protein interactions described in this article is available
via the MINT article ID
MINT-8145255
Abbreviations
DAG, diacylglycerol; DOG, dioctanoylglycerol; eEF1A1, eukaryotic elongation factor 1A1; GST, glutathione S-transferase; MEF, mouse
embryonic fibroblast; mTOR, mammalian target of rapamycin; p70S6K, p70 S6 kinase; PA, phosphatidic acid; PAS, Per-Arnt-Sim; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PK, protein kinase; PL, phospholipase; PS, phosphatidylserine; PSK, protein Ser ⁄ Thr
kinase; PtdIns, phosphatidylinositol; S6K, S6 kinase; TOP, terminal oligopyrimidine.
FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS 1757
Introduction
In lower organisms, the Per-Arnt-Sim (PAS) domain is
found often in environmental protein sensors involved
in the perception of light intensity, oxygen partial pres-
sure, redox potentials, voltage and certain ligands [1].
In mammals, the PAS domain is mainly found as a
heterodimerization interface of transcription factors

the AMP-dependent kinase homologue, sucrose
nonfermenting 1. Although PSK2 was predominantly
activated by Wsc1, PSK1 was indispensable for func-
tioning of sucrose nonfermenting 1 [10].
In mammals, PASKIN-dependent phosphorylation
inhibits the activity of glycogen synthase [11]. PASKIN
has also been suggested to be required for glucose-
dependent transcriptional induction of preproinsulin
gene expression, which might be related to PASKIN-
dependent regulation of the nuclear import of pancre-
atic duodenal homeobox-1 transcription factor [12,13].
However, by generating PASKIN-deficient knockout
mice, we could not demonstrate any PASKIN-depen-
dent difference in insulin gene expression or glucose
tolerance [14,15]. Moreover, conflicting data were also
reported on the resistance of these Paskin knockout
mice towards high fat diet-induced metabolic
syndrome [16,17].
We previously found that the eukaryotic elongation
factor 1A1 (eEF1A1) is phosphorylated by PASKIN
at T432 [18]. However, the role of this modification in
translational control awaits further investigation. In
the present study, by screening for new PASKIN
kinase targets, we demonstrate that another crucial
translation factor, ribosomal protein S6, can be phos-
phorylated by PASKIN, suggesting that PASKIN
regulates protein translation not only in yeast, but also
in mammals. Moreover, we identified phospholipid
ligands binding to PASKIN and studied their effects
on PASKIN activity.

from the same site but from distinct species. Seventeen
different pyruvate kinase-derived peptides, for exam-
ple, were identified in this way. One of the proteins
Targets and stimulation of PASKIN P. Schla
¨
fli et al.
1758 FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS
listed in Fig. 1B is glycogen synthase, which has previ-
ously been identified as a PASKIN kinase target [11].
Thus, glycogen synthase identification confirmed the
feasibility of our approach and was used as a reference
target protein for subsequent experiments.
To corroborate PASKIN-dependent phosphoryla-
tion of these rather short arrayed peptides, 11 of the
most strongly phosphorylated candidate PASKIN
kinase targets were synthesized as 20-mer peptides and
used for in vitro phosphorylation by recombinant
PASKIN (Table S2). As shown in Fig. 1C, six peptides
were significantly better phosphorylated by PASKIN
than the unrelated control peptide, and three of them
showed an even stronger phosphorylation than the
known PASKIN targets glycogen synthase and pan-
creatic duodenal homeobox-1 (i.e. 40S ribosomal
protein S6, phosphorylase kinase b and 6-phospho-
fructo-2-kinase ⁄ fructose 2,6-bisphosphatase).
Ribosomal protein S6 is phosphorylated by
PASKIN
Because a role for PASKIN in protein translation has
been reported previously [8,18], the finding that a
R

translation by p70 S6 kinase (p70S6K)-mediated phos-
phorylation of S6 at Ser235 ⁄ 236 [20]. Therefore,
recombinant S6 was expressed and purified either as
wild-type, C-terminally truncated or Ser235 ⁄ 236Ala
double-mutant glutathione S-transferase (GST) fusion
protein (Fig . 2A). As shown in Fig. 2B, PASKIN
phosphorylated wild-type but not truncated or serine
double-mutant S6 in vitro, suggesting that PASKIN
also targets S6 at Ser235 ⁄ 236.
To analyze PASKIN-dependent phosphorylation of
endogenous S6 in vivo, we used mouse embryonic
fibroblasts (MEFs) derived from either Paskin
+ ⁄ +
wild-type or Paskin
) ⁄ )
knockout mice [14]. However,
as shown in Fig. 2C, no difference in constitutive
p70S6K or S6 phosphorylation could be detected in
these cells. Because basal S6 phosphorylation by
p70S6K might overcome subtle changes caused by
PASKIN, we next used MEFs deficient for both genes
encoding mouse p70S6K (S6K1
) ⁄ )
⁄ S6K2
) ⁄ )
) [21], and
transiently overexpressed full-length PASKIN or an
N-terminally truncated version preserving the kinase
domain in these cells. Whereas S6 total protein levels
remained unchanged, phosphorylated S6 was strongly

Fig. 2. Ribosomal protein S6 is phosphory-
lated by PASKIN. (A) Sequence comparison
of the S6 peptides used in the microarray,
20-mer peptide used for the in vitro reac-
tions, and recombinant GST fusion proteins
purified from E. coli. (B) Phosphorylation
reactions in vitro using purified His
6
-PASKIN
and recombinant S6 in the presence of
[c-
33
P]ATP. Subsequent to SDS ⁄ PAGE, the
phosphorylated proteins were visualized by
phosphorimaging. Equal input was con-
trolled by immunoblotting against S6 and
the GST-tag. (C) Immunoblot analysis of the
phosphorylation status of p70S6K and S6 in
Paskin
+ ⁄ +
and Paskin
) ⁄ )
MEFs. (D) Immu-
noblot analysis of the phosphorylation status
of p70S6K and S6 in S6K1
) ⁄ )
⁄ S6K2
) ⁄ )
dou-
ble-knockout MEFs after overexpression of

33
P]ATP. Subsequent to SDS ⁄ PAGE, the phosphorylated
proteins were visualized by phosphorimaging. (B, C) Stimulation of PASKIN and PKCd autophosphorylation by increasing amounts of the
indicated phospholipids. Subsequent to SDS ⁄ PAGE, the phosphorylated proteins were visualized (upper panels) and quantified (lower panels)
by phosphorimaging. The values were normalized to 100 lgÆmL
)1
PS and 10 lgÆmL
)1
DOG ⁄ 100 lgÆmL
)1
PS mixtures for PASKIN and PKCd,
respectively (filled columns). (D) PLD but not PLC converts PC from a low affinity to a high affinity PASKIN ligand. Ninety-six-well plates
were coated with increasing amounts of PC, followed by treatment with PLD or PLC, as indicated. Binding of 100 ng of PASKIN added to
each well was detected by ELISA. Mean ± SD values of a representative experiment performed in triplicate are shown.
P. Schla
¨
fli et al. Targets and stimulation of PASKIN
FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS 1761
phospholipids had only marginal effects on PKCd.As
shown previously [24], a mixture between DOG and
PS was required to maximally induce PKCd activity.
However, combining DOG with phospholipids did not
further induce PASKIN (data not shown).
The rather unselective stimulation of PASKIN activ-
ity by all tested phospholipids suggested that the core
phospholipid moiety might confer PASKIN binding.
Indeed, as shown in Fig. 3C, phosphatidic acid (PA)
alone was sufficient to stimulate PASKIN autophos-
phorylation. The finding that PA but not DOG
strongly bound PASKIN suggested that phospholipase

2
and
PtdIns(3,4,5)P
3
, respectively (Fig. 4A). This finding
was corroborated by using dot blots with increasing
amounts of all possible PtdIns-phosphates: PASKIN
dose-dependently bound PtdIns-monophosphates bet-
ter than PtdIns-diphosphates, and nonphosphorylated
or tri-phosphorylated PtdIns bound PASKIN only
weakly (Fig. 4B, left). Similar results were obtained
with autophosphorylated PASKIN (Fig. 4B, right),
suggesting that PASKIN phosphorylation status does
not interfere with selective PtdIns-monophosphate
binding.
To localize the region responsible for PtdIns-mono-
phosphate binding, four different fragments of
PASKIN (Fig. 4C, left) were expressed and purified as
His
6
-tagged fusion proteins. However, only the kinase
domain of PASKIN bound PtdIns-monophosphates
(Fig. 4C, right), rather than the previously suggested
ligand-binding PAS domain (data not shown). We next
aimed to determine the effects of differently phosphor-
ylated PtdIns on PASKIN autophosphorylation. As
shown in Fig. 4D, autophosphorylation was dose-
dependently enhanced by all three PtdIns-monophos-
phates, whereas especially high concentrations of
PtdIns(4,5)P

However, nonphosphorylated PtdIns did not signifi-
cantly change the target phosphorylation efficiency.
Discussion
In the present study, we identified various novel poten-
tial PASKIN substrates by peptide microarray phos-
phorylation, including glycogen synthase that was
known before to be phosphorylated by PASKIN [11].
Thus, the repetitive identification of this PASKIN
target confirms, at least partially, the validity of the
peptide array approach. Other peptides derived from
proteins involved in glycogen metabolism included
Targets and stimulation of PASKIN P. Schla
¨
fli et al.
1762 FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS
phosphorylase kinase, inhibitor of protein phosphatase
1 and yeast glycogen phosphorylase (Table S1). The
involvement of PASKIN in the regulation of glycogen
synthesis was demonstrated previously by showing that
both mammalian and yeast glycogen synthases, as well
as yeast UDP-glucose pyrophosphorylase, are known
phosphorylation targets of mammlian PASKIN and
yeast PSK1 and PSK2, respectively [8,11]. However,
although Ser640 was the main PASKIN kinase target
residue of mammalian glycogen synthase [11], the pep-
tides phosphorylated by PASKIN on the microarray
contained Ser3 and Ser7 but not Ser640. Of note, a
Ser640Ala mutation did not completely prevent phos-
phorylation [11]. Therefore, our data suggest that
PASKIN might phosphorylate Ser3 and ⁄ or Ser7 of

allowed to bind to the indicated lipids
immobilized on a membrane, and subse-
quently detected using PASKIN antibodies.
(B) PASKIN dose-dependently bound
preferably PtdIns-monophosphates. PASKIN
was either detected by immunoblotting (left
panel) or by phosphorimaging after
autophosphorylation in the presence of
[c-
33
P]ATP (right panel). (C) Fragments of
PASKIN were expressed in E. coli and
purified as His
6
-tagged fusion proteins (left
panel). Subsequent to binding to the lipid
dot blots and detection using a His-tag anti-
body, only the kinase (KIN) domain of
PASKIN was found to interact with
PtdIns-monophosphates (right panel). (D)
His
6
-PASKIN autophosphorylation was
mainly stimulated by the presence of the
PtdIns-monophosphates. In vitro phosphory-
lation reactions in the presence of
[c-
33
P]ATP and the indicated synthetic diC8
PtdIns (3.16 l

phates (100 l
M) as indicated. Subsequent to
separation by SDS ⁄ PAGE, protein phosphor-
ylation was viusalized (left panel, represen-
tative images) and quantified (right panel) by
phosphorimaging. His
6
-PASKIN autophos-
phorylation without lipid and target (first lane
from the left) was used for intra-assay nor-
malization of the values. Columns represent
the mean ± SD values of three independent
experiments (*P < 0.05; **P < 0.01;
***P < 0.001; t-test).
Targets and stimulation of PASKIN P. Schla
¨
fli et al.
1764 FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS
serines of S6, starting with S236 and S235 (i.e. the
same sites as shown in the present study for PASKIN)
followed by S240, S244 and S247 [25]. A second family
of S6 kinases are p90 ribosomal S6 kinases that phos-
phorylate S6 upon mitogenic stimulation at the same
sites as PASKIN [22]. Phosphorylation of S6 by
p70S6K has long been considered to increase protein
translation by selectively enhancing the translation of
5¢-terminal oligopyrimidine (TOP) mRNAs, a subset
of mRNAs containing an oligopyrimidine tract in their
5¢-UTRs. Of note, the 5¢-TOP mRNAs code for ribo-
somal proteins and translation factors, including

PASKIN is a target of intracellular PLD cell signalling.
Unexpectedly, PtdIns-monophosphates were found
to be the best ligands of PASKIN, with clearly higher
affinities than PtdIns-diphosphates or PtdIns-triphos-
phate. PtdIns-binding domains have been reported to
display either well-defined 3D folds [29], or rather
unstructured regions with basic (for binding of the
phosphate groups) and hydrophobic residues, such as
in the noncanonical pleckstrin homology domain of
Tiam1 [30]. We identified a lysine rich region, spanning
from Lys1019 to Lys1034 of PASKIN, which shares
characteristic features with noncanonical pleckstrin
homology domains, including a double-lysine motif
(Lys1031 ⁄ 1032). However, mutation and deletion anal-
yses of this putative binding region did not affect lipid
binding by PASKIN (data not shown). Thus, it is diffi-
cult to predict the PtdIns-monophosphate binding site
within the PASKIN kinase domain and further work
will be necessary to identify the specific residues
involved in lipid binding.
Although PtdIns(4,5)P
2
and PtdIns(3,4,5)P
3
are
involved in signalling processes at the plasma mem-
brane, PtdIns-monophosphates are more abundant in
intracellular membrane structures such as the Golgi
apparatus and endosomes [31]. Within these structures,
PtdIns-monophosphates are involved in sorting and

have obtained the first indication of the upstream regu-
lators of PASKIN activity. It will be interesting to
examine how these regulators affect the downstream
processes mediated by PASKIN.
Experimental procedures
Plasmids
All cloning work was carried out using Gateway technology
(Invitrogen, Carlsbad, CA, USA). The human PASKIN
P. Schla
¨
fli et al. Targets and stimulation of PASKIN
FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS 1765
cDNA containing plasmids pENTR4-hPASK and pENTR4-
hKIN, as well as plasmids for recombinant expression of full
length His
6
-PASKIN, PASKIN truncations and eukaryotic
elongation factor 1A1, have been reported previously [18].
pENTR4-hPASK and pENTR4-hKIN were recombined
into pcDNA3.1 ⁄ c-myc-DEST [32] using LR recombinase
(Invitrogen) to generate pcDNA3.1 ⁄ c-myc-hPASK and
pcDNA3.1 ⁄ c-myc-hKIN for c-myc-tagged expression of
PASKIN or its kinase domain, respectively, in mammalian
cells. Human ribosomal protein S6 (IRAUp969B0849D6;
Deutsches Ressourcenzentrum fu
¨
r Genomforschung, Berlin,
Germany) was cloned into pENTR4 using primers 5¢-
TTATGTCGACATGAAGCTGAACAT-3¢ (forward) and
5¢-TACGTGCGGCCGCTTATTTCTGACTGGATTCAGA

or without 2 lg of recombinant target proteins in kinase
buffer (25 mm Tris–HCl, pH 7.5, 10 mm MgCl
2
,1mm
dithiothreitol) for 20 min in the presence of 3 lCi
[c-
33
P]ATP (Hartmann Analytic, Brunswick, Germany).
Proteins were separated by SDS ⁄ PAGE and analyzed by
phosphorimaging of the dried gels (Molecular Imager FX;
Bio-Rad) using quantity one software (Bio-Rad). Lipids
(Sigma, St Louis, MO, USA or Fluka, Buchs, Switzerland)
were dissolved in CHCl
3
, aliquotted in test tubes and the
CHCl
3
evaporated under a stream of nitrogen. Lipids were
then resuspended in kinase assay master mixes by thorough
vortexing. PtdIns present in the phosphorylation reactions
were obtained from Echelon Biosciences (Salt Lake City,
UT, USA) as synthetic diC8-lipids and added to the reac-
tions from 1 mm aequous stock solutions to the final con-
centrations indicated.
Peptide microarrays
Peptide microarrays were phosphorylated with recombinant
PASKIN in accordance with the manufacturer’s instruc-
tions (Pepscan, Lelystad, The Netherlands). In brief, 50 lL
of a solution containing 500 ng recombinant PASKIN,
50 mm Hepes (pH 7.4), 20 mm MgCl

with 500 lL of 100 mm Tris (pH 8.0). Phosphorylation of
the beads was quantified by liquid scintillation counting
(Packard Tri-Carb 2900TR; Perkin Elmer, Boston, MA,
USA).
Cell culture, transfections and immunoblotting
MEF cells were generated from Paskin
+ ⁄ +
and Pa-
skin
) ⁄ )
mice [14] at embryonic day 14. S6K1
) ⁄ )
⁄ S6K2
) ⁄ )
double-knockout MEFs were kindly provided by G. Tho-
mas and S. C. Kozma (Friedrich Miescher Institute for
Biomedical Research, Basel, Switzerland). MEF cells were
cultivated in DMEM (Sigma) supplemented with 10%
fetal bovine serum (Invitrogen) up to passage 12, suggest-
ing that they immortalized spontaneously. MEFs were
transiently transfected using Lipofectamine 2000 (Invitro-
gen) in accordance with the manufacturer’s instructions.
Thirty-six hours post-transfection, cells were harvested
and whole cell lysates were generated by heating the cells in
1% SDS for 5 min at 95 °C. After SDS ⁄ PAGE and
Targets and stimulation of PASKIN P. Schla
¨
fli et al.
1766 FEBS Journal 278 (2011) 1757–1768 ª 2011 The Authors Journal compilation ª 2011 FEBS
immunoblotting, the primary antibodies used were: human

4
(final concentration of 1 m) and A
450
was
determined using a microplate reader (Digiscan; Asys
Hitech, Eugendorf, Austria). For PL experiments, phospho-
lipid-coated 96-well plates were treated with 0.2 units of
PLC or PLD (Sigma), diluted in reaction buffer (120 m m
CaCl
2
, 300 mm sodium acetate, pH 5.6) for 1 h at room
temperature.
Lipid binding arrays
Membranes spotted with phospholipids were obtained from
Echelon Biosciences (P-6002, P-6100) and used in accor-
dance with the manufacturer’s instructions. Generally, 1 lg
of protein diluted in 1% skimmed dry milk in NaCl ⁄ Tris
was allowed to bind to spotted phospholipids for 16–20 h
at 4 °C. Binding was detected using primary antibodies as
indicated and horseradish peroxidase-coupled secondary
antibodies for enhanced chemiluminescence detection
(Pierce).
Acknowledgements
The authors wish to thank J. Rutter, G. Thomas and
S. C. Kozma for the generous gifts of plasmids and
cell lines, as well as Gieri Camenisch and Daniel
P. Stiehl for helpful discussions. This work was
supported by by grants from the Wolfermann-Na
¨
geli

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Supporting information
The following supplementary material is available:
Table S1. Rank order of the 75 most strongly phos-
phorylated PASKIN kinase targets on the peptide
microarray.
Table S2. Sequences of the eleven biotinylated peptides
tested for phosphorylation by recombinant PASKIN.
This supplementary material can be found in the
online version of this article.
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